The invention relates to a method of improving baseline stability in liquid chromatographic measurements and to a liquid chromatography apparatus having means for reducing baseline instabilities.
High performance liquid chromatography is a separation technique wherein a mixture of sample substances dissolved in a solvent (mobile phase) is forced under high pressure through a separation column. Different sample substances interact differently with the stationary phase in the column and therefore elute from the column at different times. The separated sample substances leaving the column are detected with a suitable detector, such as an absorbance detector, refractive index detector, conductivity detector, or electrochemical detector. The representation of the detector signal as a function of time is called chromatogram. Different sample substances are represented by individual peaks in the chromatogram; the peak area is a measure of the amount of the corresponding sample substance.
In order to ensure reproducible measurements, in particular quantitative measurements, it is important that the baseline in the chromatogram remains stable, i.e. that it is not affected by influences which have nothing to do with the sample substances. A varying baseline would interfere with the chromatographic peaks and thus lead to incorrect measuring results. In particular refractive index detectors are very sensitive to instrumental and external influences leading to unwanted baseline variations. For example, high pressure pumps forcing the solvent and sample through the column often generate pressure pulsations which can lead to refractive index signal changes which in turn are causing artefacts in the measuring signal.
A new type of baseline instability has been observed by the inventor of the present invention, in particular in connection with stationary phases comprising amino functional groups. Columns of this type are used, for example, for sugar analysis. The observed baseline instabilities occur in the form of xe2x80x9cwanderxe2x80x9d in the time range of 1-10 minutes and can thus easily interfere with chromatographic peaks.
It is thus an object of the present invention to provide a method and a corresponding apparatus for reducing the mentioned baseline variations.
It is a further object of the invention to provide a liquid chromatography apparatus having improved baseline stability.
According to the invention, these objects are achieved by for a method by claim 1 and for an apparatus by claim 9.
The invention according to claim 1 is based on the surprising finding that the baseline variation originates from variations in the adsorption of water (or one of the eluent components) to the stationary phase and that this adsorption is sensitive to temperature fluctuations. Thus, the amount of water in the mobile phase reaching the detector varies with temperature changes, leading to the baseline wander in the detector signal. The baseline problems are substantially reduced by coupling an additional solid body or liquid bath having a high heat capacity and heat conductance to the column and to an inlet capillary of the column such that the inlet capillary and the column are entirely enclosed by said solid body or liquid bath. The invention is particularly useful in connection with refractive index detection which is very sensitive to changes in the measuring conditions, but it is also advantageous for other detection methods, such as absorbance detection. It is understood that the invention is not only applicable for stationary phases with amino groups, but also for other types of stationary phases, e.g. reversed phases (RP) in connection with mobile phases containing UV absorbing additives which have a temperature dependent affinity to the stationary phase.
The invention also provides a general solution for temperature stabilization of the column, allowing a constancy of the column temperature of better than 0.001 degrees centigrade per minute. According to claim 9, an additional solid body or liquid bath having a high heat capacity and heat conductance, is coupled to the column and to an inlet capillary of the column such that the inlet capillary and the column are enclosed by said solid body or liquid bath. Preferably, the additional solid body and the inlet capillary are arranged in a thermostatted column compartment of the chromatograph which comprises a controlled heat source and/or heat sink. According to a preferred embodiment, the heat impedance between the additional solid body and the controlled heat source/sink is optimized to ensure that equilibration times do not become too long, and to avoid cross-talk of the small, fast fluctuations from the heat source/sink which would otherwise directly influence the column temperature.